Despite significant advances in the fields of cell biology, microfluidics, engineering, and three-dimensional printing, to date, conventional approaches have failed to re-create functional capillaries that feed and support the thick tissue necessary to construct a human organ. To date, these approaches in tissue engineering have relied on the in-growth of blood vessels into tissue-engineered devices to achieve permanent vascularization. This strategy has worked for some tissues that are either very thin such as a bladder wall replacement or tissues such as bone replacements that do not require vasculature to function. However, current tissue engineering techniques fall short in the creation of complex tissues such as large vital organs, including liver, kidney, thick skin, and heart. Larger tissues may also be thought of as an organization of smaller tissue sub-units; for example, the kidney is comprised of hundreds of thousands of nephron units, the functional unit of the lungs, i.e., the alveolar spaces, have a combined surface area of 70 to 80 meters squared (m2), but are only 1 cell wall, 5 to 10 micrometers (μm), thick. Current tissue printing methodology not only fails to re-create the fine microvasculature necessary to support tissues thicker than 300 micrometers (μm), but cannot organize cells into the structural orientations and niches that are necessary for organ function.
Antibodies are proteins produced and secreted by B cells during an immune response. Antibodies may have high binding specificity and affinity to potential infectious agents and thus may be used to bind to and isolate, neutralize, or alter the effects of other proteins, viruses, bacteria, chemical-protein combinations, or carbohydrate molecules. This makes antibodies a valuable protein in protection from pathogens and isolation or neutralization of infectious or otherwise pathologic agents or proteins. In addition, antibodies may be used to redirect immune responses, by modulation through either disruption or enhancement of other protein-protein interactions, by opsonization of phagocytosis, substantially increasing the likelihood of immune recognition and destruction of pathogenic or pathologic agents.
For a B cell to produce a high affinity or high avidity antibody, a multi-step process called affinity maturation is required. During affinity maturation, genetic changes in the B cell receptor (BCR) occur. Following these genetic changes, a guess-and-check, evolutionary-like process occurs. This is a competitive process in which high binding strength leads to more contact with accessory cells that give positive survival signals. Accessory cells include, but are not limited to: T cells, B cells, monocytes, macrophages, dendritic cells, natural killer cells, etc. A null BCR rearrangement ends the process of affinity maturation, and it no longer receives survival signals from accessory cells that are presenting the antigen or cross-linking of its own BCR that can provide additional positive feedback. Therefore, a higher affinity BCR rearrangement and random mutation give the B cell positive feedback, encouraging B cell division, more receptor rearrangement, and more random mutation, while a lower affinity BCR rearrangement may result in cell death or anergy. After several rounds of selection in this guess-and-check sequence, a high affinity B cell differentially survives and transitions into a plasma cell. Plasma cells circulate in the blood stream and secrete high amounts of antibody to assist an immune response. Affinity maturation occurs mostly in lymph node organs over the period of several days.
The lymph node consists of a large collection of immune cells, primarily B cells, T cells, and follicular dendritic cells (FDCs) within a reticular network. Lymph nodes enable the widespread intercellular interaction required for a full-scale immune response by increasing the proximity of cells to one another. B cell receptor rearrangement is supported by secondary survival signals from accessory cells. These proximity-based cellular interactions require or are significantly improved by a particular three-dimensional (3D) spatiotemporal arrangement of immune cells, found within the lymph node.
Three-dimensional cell movement and spatiotemporal arrangement of cells is critical for several cell-based processes, including cell differentiation and cellular responses to external or internal stimuli. During affinity maturation of B cells, the immune cells involved physically compartmentalize in the lymph node into regions that contain dividing B cells (i.e., dark zone), non-dividing B cells (i.e., light zone), and supporting accessory cells, as shown in FIG. 16. Compartmentalization of the immune cells, followed by rearrangement during activation, indicates a dependence upon this organization for the proper development of high-affinity antibodies. While B cells undergo affinity maturation, they move between compartments in the lymph node, crawling across other cells and collagen networks, as shown in FIG. 17. This disclosure describes a non-toxic, printing process of cell-containing collagen networks at a millimeter, micron, or sub-micron resolution such that a lymphoid organ or organoid containing other cell types with finite cell compartmentalization may be created for purposes including, but not limited to, antibody generation.
Development of an antigen-specific antibody in a synthetic tissue de novo after antigen challenge or vaccination of the organoid indicates functional cell-cell interactions and a functionally responsive tissue that can support complex cell-cell interactions over the course of days to weeks to months.
Antibodies have been leveraged for therapeutic purposes owing to their high efficacy and versatility in targeting, neutralizing, and/or opsonizing biological agents relevant to a number of disease states including cancer, autoimmune disease, and infectious disease. However, current methods for the discovery and production of antibodies for therapeutic or research uses are time-consuming and costly. The standard method of antibody production requires the use of animals, often mice or other rodents, rabbits, chickens, horses, or non-human primates, which are injected with an antigen and exsanguinated for B cell collection after exhibiting an immune response. Antibodies produced by this method that are intended for use against human targets (e.g., for therapeutic purposes) require an additionally laborious humanization step, which may change the binding affinity of the target, while providing no guarantee of safety or efficacy in humans. Other methods, such as phage display, use a predefined antibody library or set of sequences coupled with some method of selection for the protein of interest. Often these libraries do not yield unique or high-affinity sequences. Furthermore, as a pre-defined group of proteins, they may not yield the ability to respond to a novel infectious agent. Our technology solves the dual problems of (a) the reliance on animal models for antibody discovery and (b) the inability to produce high-affinity, unique antibodies using a high-throughput model derived from humans.
The described method involves the use of a light source, including, but not limited to, white light, blue light, green light, and single- or multi-photon laser sources of any wavelength. Light may be projected in two or three dimensions.
Two-dimensional (2D) projection is achieved by two-dimensional projection of a single axial plane with a digital micromirror device (DMD) or spatial light modulator (SLM) that has light placed only in specific regions where polymerization of a material is desired.
Three-dimensional projection, if used, may be achieved by holographic projection of light through use of a two light modulating systems in series, as disclosed in commonly invented U.S. Provisional Patent Appl. No. 62/469,948, entitled MULTI-PHOTON TISSUE PRINTING, which is incorporated herein by reference. Polymerization of biomaterials has been described and implemented for use in bioprinting of materials for cell scaffolds. The method described herein involves projecting a light source into a bath containing polymerizable material to encapsulate cells as polymerization occurs. By comparison, alternate in-media polymerization-based tissue engineering approaches use light projection to produce a 3D scaffold that may later be seeded with cells. Encapsulating cells during the polymerization process rather than seeding increases the precision with which cells may be placed; resolution that may be achieved within the polymerized space is further increased by using a two-photon light source rather than a standard single-photon light source. The use of two-photon light sources to induce polymerization both eliminates or substantially reduces the toxicity of light to cells and speeds printing to improve cell viability and growth. Multiphoton and single-photon laser methods are superior to extrusion printing in terms of resolution that may be attained and speed at which large or complex structures may be printed. Alternatively, photons of longer wavelength may be used to reduce damage to cells, and/or less intense light or shorter light exposure time may be used. Additionally, printing simultaneously in three-dimensions by holographic projection of the light source in the desired polymerization pattern substantially reduces print time, also reducing stress to cells as a result of light exposure or time outside of an incubator.
The most commonly used medical devices for wound closure, wound patching as in a stent, knitting, or fusing of tissues including bone and skin, are created from biologically inert materials. Many of these materials may dissolve over time, but many remain permanent features for many years after surgery and may induce complications or hinder the healing process.
Some more advanced materials and medical devices used for surgical wound closure or tissue repair are cell-seeded after three-dimensional extrusion printing to introduce stem cells or other cell types that might be beneficial to wound healing or closure. However, cell seeding into biologically inert materials have low viability and low survival profiles for cells and thus, incomplete delivery of beneficial cells.
Tissue implants for the promotion of tissue healing or improvement of function are often in the form of cell suspension injections or small devices that do not breach the 200-300 micrometer limit of diffusion for oxygen, nutrients and waste products, or are mostly a-cellular. Furthermore, tissue implants do not contain cells printed in place that may remodel the print material and grow within the printed material, a significant hindrance to the development of a functional tissue insert that may incorporate into the implant environment.
The engineering of medical devices that contain cells able to remodel and growth within the implanted device are limited by print resolution, lack of structurally resilient biomaterials that may be used in extrusion printing, cytotoxicity of high-resolution extrusion printing, and techniques to introduce cells into the 3D printed medical devices after printing. In addition, 3-dimensional extrusion printing of high-complexity devices is slow, often taking hours or days to complete a single print cycle. This makes production and scale-up of on-demand cell-containing devices difficult to achieve.
This disclosure describes the development and use of three-dimensional lithography enabled by holographic light projection using a technique called optical wave-front shaping for the purpose of bioprinting cell containing structures and materials. The cell containing structures and materials are designed specifically to maintain structural properties such as tensile strength, shear and compression force resistance, compressibility or other properties that allow for compatibility with surgical techniques, specifications, and native tissue and organ structures while being fully biologically compatible. Hardening or polymerization of the biomaterials may be actuated by light or laser interactions with the printing materials at specific points in three dimensional space. Printing materials include both biomaterials that are monomeric and doping or actuating agents that are non-cytotoxic but react to light or specific wavelengths of light. Biologically compatible devices or structures printed containing embedded or trapped cells allow for remodeling and break-down or resorption of the implanted device that is used to deliver cells to the desired site for the purpose of, though not limited to, healing or augmentation, or replacement of tissue function.